AB0905 DEVELOPMENT OF A NEW COMPREHENSIVE ASSAY KIT FOR MYOSITIS-ASSOCIATED ANTIBODIES AND MYOSITIS-SPECIFIC ANTIBODIES EXAMINATION OF CLINICAL USEFULNESS IN INFLAMMATORY MUSCLE DISEASES
Authors: T. Hashimoto, H. Yasuoka
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Inflammatory muscle diseases are systemic inflammatory disorders that affect not only muscles but also skin, joints, lungs, and heart. Dermatomyositis (DM) and polymyositis (PM) are characterized by the production of antibodies. Measurement of these antibodies is useful for diagnosis, classification of disease type, evaluation of disease activity, prognosis prediction, and determination of therapeutic strategy. However, the sensitivity and specificity vary depending on the measurement method, and some autoantibodies can only be measured in limited laboratories. <h3>Objectives</h3> We developed a novel comprehensive assay kit for myositis-associated antibodies with the aim of developing a rapid and simple detection method for myositis-specific or related autoantibodies. we used a proteome-wide antibody screening and quantification with wet protein arrays consisting of proteins synthesized from proteome-wide human cDNA library (HuPEX). We examined whether this assay kit is clinically useful compared to enzyme-linked immunosolbent assay (ELISA). <h3>Methods</h3> Sera from 47 patients diagnosed or suspected of inflammatory muscle disease were used to measure myositis-specific autoantibodies (anti-ARS, anti-MDA5, anti-TIF1-γ, anti-Mi-2, and anti-SRP, antibodies) and myositis-related autoantibodies (anti-U1RNP, anti-Ku, and anti-PM-Scl, anti-mitochondria antibodies) were measured using a proteome-wide antibody screening and quantification with wet protein arrays consisting of proteins synthesized from proteome-wide human cDNA library (HuPEX) and compared with the results of ELISA, which is commercially available and immunoprecipitation with some autoantibodies(anti-OJ, anti-Zo antibodies). Autoantibodies that were undetectable by the ELISA and positive only on the array were subjected to immunoprecipitation. In immunoprecipitation, primary antibody added to a solution containing an antigen to form an antigen-antibody complex in the solution. Next, a secondary antibody immobilized on the beads is added to adsorb the antigen-antibody complex to the beads. Finally, the antigen was eluted from the beads using SDS and analyzed using SDS-PAGE. <h3>Results</h3> In the 47 cases, the clinical diagnosis was DM in 26 cases, PM in 20 cases, and inclusion body myositis in 1 case. In the ELISA commercially available, 20 cases of anti-ARS antibody (17 cases of DM, 3 cases of PM), 5 cases of anti-MDA5 antibody (Amyopathic DM), 1 case of anti-TIF1-γ antibody (DM), 2 cases of Mi-2 antibody (1 case of DM, 1 case of PM), 2 cases of anti-RNP antibody (PM), and 2 cases of anti-mitochondria antibody (PM). Compared with the results measured by the ELISA commercially available, eighteen of the 20 cases were positive for anti-ARS antibodies using the proteome-wide antibody screening system and all other autoantibodies were 100% concordant. In addition, 1 case of DM in which autoantibodies could not be detected by the ELISA was positive for anti-OJ antibody by the proteome-wide antibody screening system. Of the 6 PM cases whose autoantibodies could not be detected by the ELISA, 1 was anti-HMGCR antibody-positive and 1 was anti-Zo antibody-positive, consistent with the clinical diagnosis. Furthermore, among the 4 PM cases who were positive only for anti-SS-A antibody by ELISA, 2 were anti-SRP antibody positive, 1 was anti-Ku antibody positive, and 1 was anti-Ki antibody positive by the proteome-wide antibody screening system. Those samples that could be detected by the array were also confirmed to be positive by immunoprecipitation. <h3>Conclusion</h3> Collectively, proteome-wide screening of antibodies using the <i>in vitro</i> proteome can reveal the myositis-associated antibodies of patients and may provide novel clinical biomarkers. <h3>References</h3> [1]Mimori T, Autoantibodies in idiopathic inflammatory myopathy:an update on clinical and pathophysiological significance. Curr Opin Rheumatol, 2007. 19: p. 523-529 [2]Fukuda E, et al. Identification and characterization of the antigen recognized by the germ cell mAb TRA98 using a human comprehensive wet protein array. Genes Cells, 2021. 26(3): p.180-189. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.